The term epigenetics is used to define heritable changes in gene regulation or cellular phenotype that occur without alteration in DNA sequence (Bird 2007). Epigenetic analysis could help explain why cells with identical genotype could have different phenotype. Thus epigenetics is considered to be the missing chain between genotype and phenotype (Bernstein et al. 2007; Reik 2007).
The genetic material in eukaryotic cells exists as nucleic acids and protein complex, termed chromatin. The basic unit of chromatin is the nucleosome, which is composed of DNA wrapped around the octamer of histone proteins. Nucleosomes are then packed into higher-order structures and finally form chromosomes. Based on the state of condensation chromatin is identified as euchromatin (less condensed, and containing most actively transcribed genes) or heterochromatin (highly condensed and the transcriptionally silent form of chromatin). In general, epigenetic modifications regulate gene expression level through the change in chromatin condensation state. DNA methylation usually suppresses gene transcription as it induces binding of regulating proteins, which promote chromatin condensation. Histone modifications (methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation) also regulate chromatin structure and affect DNA transcription, replication, repair and recombination (Groth et al. 2007; Koch et al. 2007; Krivtsov et al. 2008). For example histone acetylation favors euchromatin formation as it impairs the DNA-histone interaction and promotes chromatin decondensation. Small and non-coding RNAs can also regulate chromatin condensation, gene transcription, and thus are also recognized as epigenetic markers (Mattick and Makunin 2006).
DNA methylation is the most popular and widely analyzed epigenetic modification, known since 1975 (Holliday and Pugh 1975; Riggs 1975). DNA in mammalian genomes is primarily methylated at CpG sites. Most of CpG sites are located in so called CpG islands. CpG islands are described as 0.5 kb-5 kb long, GC rich (>60%) genome regions with a high frequency of CpG dinucleotides. CpG islands are usually located in the vicinity of promoters or the first exons of the genes. Methylation in CpG islands usually results in transcription inactivation and gene silencing (Robertson and Wolfe 2000).
5-methyl cytosine (m5C) is the most abundant DNA modification in the mammalian genome. Often m5C is called the fifth base of DNA. Methylation of cytosine in mammals occurs within CpG sites and is catalyzed by DNA methyltransferases. De novo methylation of CpG dinucleotides is performed by DNMT3a and DNMT3b DNA methyltranferases. DNMT1 DNA methyltransferase maintains the DNA methylation pattern during the DNA replication cycle (Bird 2002). In addition to 5-methyl cytosine (m5C), one more cytosine modification, 5-hydroxymethyl cytosine (hm5C), was discovered recently in mouse brain cells (Kriaucionis and Heintz 2009). The biological significance of hm5C is still under investigation. However it is hypothesized that hm5C participates in DNA demethylation processes and regulation of gene transcription.
The most popular technique used for DNA methylation analysis is bisulfiite treatment. This process deaminates all unmodified cytosines into uracils, while m5C and hm5C are resistant to this type of conversion. During bisulfiite treatment cytosines in the target sequence are changed to uracil if they are umethylated, or remain unchanged if they are methylated. Changes in DNA sequence can further be detected using molecular biology techniques such as DNA sequencing, PCR, qPCR, restriction analysis, Southern blotting, primer extension, HPLC, and MALDI-TOF MS etc. (Esteller 2008; Suzuki and Bird 2008).
Along with bisulfite analysis, DNA methylation status can be interrogated using different restriction endonucleases, whose cleavage of DNA at particular target sequences can be either blocked (Colaneri et al. 2011) or induced (Zheng et al. 2010; Cohen-Karni et al. 2011) by the presence of methylated cytosine. The level of target DNA digestion is interrogated using similar techniques as in case of bisulfite modification. Methylation status comparison between control and test genomic DNA samples can be performed employing differential methylation hybridization (DMH) technique (Huang et al. 1999). Genomic arrays of CpG islands are used to hybridize genomic DNA digested with methylation-sensitive endonucleases and amplified by PCR.
A number of restriction endonucleases sensitive to cytosine methylation are available commercially. Many are used in different techniques to assess methylation status of individual CpG targets or methylation signatures of genomic samples. For example, restriction endonucleases BstUI (CGCG), HpaII (CCGG), and HhaI (GCGC) were used in differential methylation hybridization (DMH) experiments to reveal methylation status differences between control and test samples of genomic DNA (Yan et al. 2002). Another example of methylation status analysis also employs the set of four restriction endonucleases sensitive to cytosine methylation AciI (CCGC), HpaII (CCGG), HinP1I (GCGC), and HpyCH4IV (ACGT) and next generation sequencing approach (Colaneri et al. 2011). In both examples the authors have used only methylation sensitive restriction endonuclease digestion analysis.
It is possible to analyze the methylation level of genomic DNA with restriction enzyme isoschizomer pairs, where the enzymes in the pair have differing sensitivities to CpG methylation. To date there is only one pair of restriction endonucleases, MspI and HpaII, with these capabilities. MspI and HpaII are isoschizomers that recognize the target sequence 5′-CCGG-3′. When the internal CpG in the 5′-CCGG-3′ tetranucleotide sequence is methylated cleavage with HpaII is blocked, but cleavage with MspI is not affected. Thus parallel digestions of genomic DNA samples with MspI and HpaII can be used to determine the methylation level of the internal cytosine in the CpG base pair located in the sequence 5′-CCGG-3′ (Hatada et al. 2006; Khulan et al. 2006; Oda et al. 2009; Takamiya et al. 2009).
This method is only able to examine methylation status in the context of 5′-CCGG-3′ and some genes or gene regions of interest, e.g., AT rich regions, could have no CCGG sites. Conversely, in CpG rich sequences such as gene promoters where regions of CpG islands are located, there may be many adjacent HpaII/MspI sites, so that methylation analysis of individual CpG base pairs employing HpaII/MspI digestion and qPCR quantification could be impaired as even short qPCR amplicons could have several 5′-CCGG-3′ sequences. In particular, after restriction digestion, products are analyzed by qPCR with a primer pair flanking the CCGG site of interest. HpaII/MspI sites may be too close to each other in CpG islands to successfully design amplicon suitable for qPCR analysis; for optimal PCR efficiency a qPCR amplicon usually is about 100 bases length. Moreover, the cleavage of both HpaII and MspI is blocked by m5C modification of the outer cytosine in the 5′-CCGG-3′ recognition sequence, and in some cases this could impair the interpretation of CpG methylation status in the target sequence.
Further analysis methods involve enrichment of DNA carrying methylated cytosines from the total pool of shared or fragmented DNA using methylated DNA immunoprecipitation (MeDIP or mDIP) technique (Weber et al. 2005). Enriched methylated DNA fragments can be further analyzed using high-throughput DNA analysis methods such as DNA microarrays (MeDIP-chip) or next generation sequencing (MeDIP-seq). Sequencing is the most informative and preferred analysis technique, whether it is used in combination with bisulfite modification, methylation-sensitive DNA digestion, or DNA immunoprecipitation. Although next generation sequencing (NGS) prices remain high, continuously increasing capabilities and decreasing cost of NGS in the near future should provide an opportunity to perform whole genome or at least exome methylation analysis as a routine approach in diagnostic laboratories.
Despite the above methods, and in view of the awareness of potential importance of DNA methylation in phenotype, the need exists for further tools that can be used to analyze DNA modification status in the rapidly growing field of epigenetic research.